Method of co-culturing mammalian muscle cells and motoneurons

ABSTRACT

The invention provides a method of co-culturing mammalian muscle cells and mammalian motoneurons. The method comprises preparing one or more carriers coated with a covalently bonded monolayer of trimethoxysilylpropyl diethylenetriamine (DETA); suspending isolated fetal mammalian skeletal muscle cells in serum-free medium according to medium composition 1; suspending isolated fetal mammalian spinal motoneurons in serum-free medium according to medium composition 1; plating the suspended muscle cells onto the one or more carriers at a predetermined density and allowing the muscle cells to attach; plating the suspended motoneurons at a predetermined density onto the one or more carriers and allowing the motoneurons to attach; covering the one or more carriers with a mixture of medium composition 1 and medium composition 2; and incubating the carriers covered in the media mixture.

RELATED APPLICATION

This application claims priority from provisional application Serial No.61/171,958 which was filed on 23 Apr. 2009, and which is incorporatedherein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under R01 NS050452awarded by the National Institutes of Health. The government has certainrights in the invention.

FIELD OF THE INVENTION

The present invention relates to the field of neurobiology and, moreparticularly, to a method of inducing formation of functionalneuromuscular junctions in a co-culture of mammalian muscle cells withmammalian motoneurons which remains viable for up to approximately sevenweeks.

BACKGROUND OF THE INVENTION

The neuromuscular junction (NMJ), formed between motoneurons andskeletal muscle fibers, is one of the most studied synaptic structures(Witzemann 2006). In a mammalian vertebrate, whenever an actionpotential is fired by a motoneuron, pre-synaptic vesicles loaded withthe neurotransmitter acetylcholine (ACh) are released in the synapticcleft (Chow et al., 1985). The released ACh diffuses across the synapticcleft and binds to the post-synaptic terminals in the muscle enrichedwith receptors for acetylcholine (AChRs). This leads to musclecontraction. In this transmission process the electrical impulses(action potentials) generated by the motoneuron are converted tochemical signals, then the chemical signals are converted into amechanical signal in the form of muscle contraction. Therefore, not onlydo NMJs represent an important system for studying synapse formation andmaturation, but also for studying how cells interconvert messagesbetween electrical, chemical and mechanical modalities.

In vivo, NMJ formation is a multistep process, requiring the spatial andtemporal interaction of growth factors, hormones and cellular structuresthat results in a pre-synaptic axonal terminal interfaced with a regionof the skeletal muscle membrane (postsynaptic) prepatterned with AChRs(Colomar et al., 2004; English 2003). In vitro culture models representa powerful cell biology tool to study the role of these different growthfactors, hormones and cellular structures involved in NMJ formation in adefined, controlled system. Consequently, the development of an in vitrosystem resulting in NMJ formation would facilitate investigations intothe roles of specific factors involved in, and required for, the processto occur efficiently.

However, limited success has been achieved in developing a long-term invitro system for NMJ formation in the absence of serum containing mediaand biological substrates. These issues limit the reproducibility of invitro studies and their translation to tissue engineering applicationsand high-throughput assay development. For example, the concentrationand/or temporal application of medium components could be investigatedto determine their influence on NMJ formation, maturation andmaintenance. Such a system also benefits from the absence of factorsthat may be present in serum that would inhibit these processes.Employing a non-biological growth substrate such astrimethoxysilylpropyl diethylenetriamine (DETA) provides an additionalmeasure of control. DETA is a silane molecule that forms a covalentlybonded monolayer on glass coverslips, resulting in a uniform,hydrophilic surface for cell growth. The use of DETA surfaces isadvantageous from a tissue engineering perspective because it can becovalently linked to virtually any hydroxolated surface, it is amenableto patterning using standard photolithography (Ravenscroft et al., 1998)and it promotes long-term cell survival because it is non-digestible bymatrix metalloproteinases secreted by the cells (Das et al., 2004; Daset al., 2007 (Nat. Protocols)). It is also possible that its structuralrelationship to the growth factor spermidine, which has recently beenshown to prolong cell life (Eisenberg et al., 2009), contribute to itsunique ability to enable long-term healthy cell cultures.

Previously, we developed a defined in vitro model facilitating theshort-term co-culture of motoneurons and skeletal muscle that resultedin NMJ formation (Das et al., 2007 (Neuroscience)). This model alsoutilized a biocompatible silane substrate and a serum-free mediumformulation. However, further improvements were necessary to enhance thephysiological relevance of the NMJ development system. Limitations ofthe previous model were that it did not support long-term tissueengineering studies and therefore, could not mimic several of the musclematuration processes observed in vivo by obtaining myotubes that moreaccurately represent mature extrafusal fibers.

As noted, neuromuscular junction (NMJ) formation, occurring betweenmotoneurons and skeletal muscle, is a complex multistep processinvolving a variety of signaling molecules and pathways. In vitromotoneuron-muscle co-cultures are powerful tools to study the role ofdifferent growth factors, hormones and cellular structures involved inNMJ formation. In this study we have demonstrated a co-culture systemthat enable sarcomere assembly in the skeletal muscle myotubes asevidenced by A band/I band formation, increased NMJ density andselective myosin heavy chain (MHC) class switching. These resultssuggest we have discovered a group of biomolecules that act as molecularswitches promoting NMJ formation and maturation as well as skeletalmuscle fiber maturation to the extrafusal phenotype. This model systemwill be a powerful tool in basic NMJ research, tissue engineered NMJsystems, bio-hybrid device development for limb prosthesis and inregenerative medicine. It could also be useful in new screeningmodalities for drug development and toxicology investigations.

SUMMARY OF THE INVENTION

With the foregoing in mind, the present invention advantageouslyprovides a serum-free culture system utilizing defined temporal growthfactor application and a non-biological substrate resulted in theformation of robust NMJs. The system resulted in long-term survival ofthe co-culture and selective expression of neonatal myosin heavy chain,a marker of myotube maturation. NMJ formation was verified bycolocalization of dense clusters of acetylcholine receptors visualizedusing alpha-bungarotoxin and synaptophysin containing vesicles presentin motoneuron axonal terminals.

This model will find applications in basic NMJ research and tissueengineering applications such as bio-hybrid device development for limbprosthesis and regenerative medicine as well as for high-throughput drugand toxin screening applications.

The present invention provides a method of co-culturing mammalian musclecells and mammalian motoneurons. The method yields functionalneuromuscular junctions in a culture which is particularly long-lived,up to approximately 7 weeks.

The method includes preparing one or more carriers coated with acovalently bonded monolayer of trimethoxysilylpropyl diethylenetriamine(DETA). The carriers are preferably glass cover slips as used formicroscopy applications. The method continues by suspending isolatedfetal mammalian skeletal muscle cells in serum-free medium according tomedium composition 1, followed by suspending isolated fetal mammalianspinal motoneurons in serum-free medium according to mediumcomposition 1. Next is plating the suspended muscle cells onto the oneor more carriers at a predetermined density and allowing the musclecells to attach and plating the suspended motoneurons at a predetermineddensity onto the one or more carriers and allowing the motoneurons toattach. The method continues by then covering the one or more carrierswith a mixture of medium composition 1 and medium composition 2 andincubating the carriers covered in the media mixture.

It is preferable that in carrying out the presently disclosed method,the practitioner verify DETA monolayer formation by one or more opticalparameters, for example, with a contact angle goniometer and by X-rayphotoelectron spectroscopy (XPS).

In the method, the mammalian skeletal muscle cells and mammalian spinalmotoneurons preferably originate from fetal rats. In this regard, whenplating the muscle cells it is preferably done at a density ofapproximately from 700 to 1000 cells/mm² and the motoneurons arepreferably plated at a density of approximately 100 cells/mm². It shouldbe understood that incubating is effected under mammalian physiologicconditions, as is known in the art for mammalian cell tissue culture.Particularly, incubating is best effected at approximately 37° C. in anair atmosphere with about 5% CO₂ and 85% humidity.

In the method, covering comprises a mixture of approximately equalvolumes of medium composition 1 and medium composition 2. A completechange of the medium covering the carriers by substituting NbActiv4medium without growth factors is preferred during the first week ofincubation and most preferred on day 4 of incubation. Afterwards, themethod calls for changing spent medium as needed with fresh NbActiv4medium without growth factors. In a preferred embodiment of the method,this periodic changing of the medium may be accomplished every 3 days.

In another embodiment of the presently disclosed method, co-culturingmammalian muscle cells and motoneurons includes allowing fetal musclecells and fetal spinal motoneurons suspended in a serum-free mediumaccording to composition 1 to adhere to a monolayer of covalently bondedDETA supported on an underlying carrier surface. Following this, themethod calls for incubating the adhered muscle cells and motoneuronscovered in a mixture of serum-free medium composition 1 and serum-freemedium composition 2. Further details of this alternate embodiment areas noted above.

Yet another embodiment or variation of the present invention includes amethod of inducing in vitro formation of functional neuromuscularjunctions. This embodiment includes depositing a suspension of isolatedfetal muscle cells and fetal spinal motoneurons in a mixture of mediumcompositions 1 and 2 onto a film of DETA supported on a carrier surface,allowing the cells to adhere to the film and culturing the cells undermammalian physiologic conditions. This is followed by changing themedium mixture to medium composition 2 without growth factors beforeseven days of culturing the cells, and exchanging spent medium duringculturing for fresh medium composition 2 without growth factors. Themethod also includes monitoring the cells while culturing for formationof functional neuromuscular junctions between motoneurons and musclecells.

BRIEF DESCRIPTION OF THE DRAWINGS

Some of the features, advantages, and benefits of the present inventionhaving been stated, others will become apparent as the descriptionproceeds when taken in conjunction with the accompanying drawings,presented for solely for exemplary purposes and not with intent to limitthe invention thereto, and in which:

FIG. 1 is a protocol for long-term NMJ formation in a motoneuron andskeletal muscle co-culture, according to an embodiment of the presentinvention;

FIG. 2 shows phase contrast micrographs of the motoneuron and skeletalmuscle co-culture between days 12-15. (A-D); red arrows indicate thedistinct morphology of the motoneuron and its processes; green arrowsindicate the myotubes; scale bar=25 μm;

FIG. 3 provides phase contrast pictures of the co-cultures between days25-30; (A, B) the myotubes exhibited characteristic striations; (C, D)myotubes with striations and myotubes without striations; red arrowsindicate the motoneuron cell body and the processes; green arrowsindicate the myotubes; scale bar for A, B=40 μm; scale bar for C, D=25μm;

FIG. 4 shows the immunocytochemistry of co-cultures at day 25; (A-B)NF-150 (red) indicates the large motoneurons and their processes (whitearrows); the striated myotubes (green) stained for nMHC(N3.36); scalebar=50 μm;

FIG. 5 depicts neuromuscular junction (NMJ) formation between day 30-40;(A) phase picture of the myotube indicating the alpha-bungarotoxinstaining in green; (B) triple stain, showing the close proximity ofalpha-bungarotoxin (green) and synaptophysin (blue) indicating synapseformation at a specific confocal plane and myotube striations areindicated in red (nMHC); (C-D) NMJ observed at two different planesusing confocal microscopy; a much more dense clustering of synaptophysinand alpha-bungarotoxin was observed in these planes;

FIG. 6 shows striated myotube development in the absence of NMJformation; (A, B) no NMJs were observed on this striated myotube; (A) aphase picture of the myotube; (B) immunostained picture of the samemyotube with alpha-bungarotoxin, N3.36 and synaptophysin; scale bar=50μm; and

FIG. 7 depicts NMJ formation on an N3.36 (−) myotube; (A) phase pictureshowing the different morphologies of myotubes in the co-culture; (B-D)show that NMJ formation was observed on a myotube that was negative forN3.36; culture stained with alpha-bungarotoxin, N3.36 and synaptophysin;possibly the myotube on which NMJ was formed was immature and had notyet expressed the neonatal myosin heavy chain marker (N3.36).

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention will now be described more fully hereinafter withreference to the accompanying drawings, in which preferred embodimentsof the invention are shown. Unless otherwise defined, all technical andscientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which this inventionpertains. Although methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, suitable methods and materials are described below. Anypublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including any definitions,will control. In addition, the materials, methods and examples given areillustrative in nature only and not intended to be limiting.Accordingly, this invention may be embodied in many different forms andshould not be construed as limited to the illustrated embodiments setforth herein. Rather, these illustrated embodiments are provided so thatthis disclosure will be thorough and complete, and will fully convey thescope of the invention to those skilled in the art. Other features andadvantages of the invention will be apparent from the following detaileddescription, and from the claims.

Materials and Methods

Surface Modification and Characterization

Glass coverslips (Thomas Scientific 6661F52, 22×22 mm No. 1) werecleaned using an O2 plasma cleaner (Harrick PDC-32G) for 20 min at 100mTorr. The DETA (United Chemical Technologies Inc. T2910KG) films wereformed by the reaction of the cleaned glass surface with a 0.1% (v/v)mixture of the organosilane in freshly distilled toluene (Fisher T2904).The DETA coated coverslips were then heated to approximately 100° C.,rinsed with toluene, reheated to approximately 100° C., and then ovendried (Das et al., 2006). Surfaces were characterized by contact anglemeasurements using an optical contact angle goniometer (KSV Instruments,Cam 200) and by X-ray photoelectron spectroscopy (XPS) (Kratos Axis165). XPS survey scans, as well as high-resolution N1s and C1s scansutilizing monochromatic AI Ka excitation were obtained (Das et al.,2006).

Skeletal Muscle Culture in Serum-Free Medium

Skeletal muscle was dissected from the thighs of the hind limbs of fetalrat (17e18 days old). Briefly, rats were anaesthetized and killed byinhalation of an excess of CO₂. This procedure was in agreement with theAnimal Research Council of University of Central Florida, which adheresto IACUC policies. The tissue was collected in a sterile 15 mLcentrifuge tube containing 1 mL phosphate-buffered saline (calcium- andmagnesium-free) (Gibco 14200075). The tissue was enzymaticallydissociated using 2 mL of 0.05% of trypsin-EDTA (Gibco 25300054)solution for 30 min in a 37° C. water bath at 50 rpm. After 30 min thetrypsin solution was removed and 4 mL Hibernate E/10% fetal bovine serum(Gibco 16000044) was added to terminate the trypsin reaction. The tissuewas then mechanically triturated with the supernatant being transferredto a 15 mL centrifuge tube. The same process was repeated two times byadding 2 mL of L15/10% FBS each time. The 6 mL cell suspension obtainedafter mechanical trituration was suspended on a 2 mL, 4% BSA (SigmaA3059) (prepared in L15 medium) cushion and centrifuged at 300 g for 10min at 4° C. The pellet obtained was washed 5 times with L15 medium thenresuspended in 10 mL of L15 and plated in 100 mm uncoated dishes for 30min. The non-attached cells were removed and then centrifuged on a 4%BSA cushion (Das et al., 2006).

The pellet was resuspended in serum-free medium according to theprotocol illustrated in FIG. 1 and plated on the coverslips at a densityof 700-1000 cells/mm². The serum-free medium containing different growthfactors and hormones was added to the culture dish after 1 h. The finalmedium was prepared by mixing medium 1 (Table 1) and medium 2 (Table 2)in a 1:1 v/v ratio. FIG. 1 indicates a flowchart of the cultureprotocol. Tables 1 and 2 list the growth factor and hormone supplementcompositions of medium one and medium two. The cells were maintained ina 5% CO₂ incubator (relative humidity 85%). The entire medium wasreplaced after four days with NbActiv4 medium according to the protocolin FIG. 1 (Brewer et al., 2008). As described in (Brewer et al., 2008),NbActiv4™ (available from BrainBits LLC) comprises all of theingredients in Neurobasal™, B27™, and Glutamax™. NbActiv™ may alsocomprise creatine, estrogen, and cholesterol. Thereafter three-fourthsof the medium was changed every three days with NbActiv4.

TABLE 1 Composition of Medium 1 No. Component Amount Catalogue # SourceReferences 1 Neurobasal A 500 mL 10888 Gibco/Invitrogen Brewer et al.,1993 2 Antibiotic-Antimycotic 5 mL 15240-062 Gibco/Invitrogen 3 Glutamax5 mL 35050-061 Gibco/Invitrogen 4 B27 Supplement 10 mL 17504-044Gibco/Invitrogen Das et al., 2004; Brewer et al., 1993 5 G5 Supplement(100X) 5 mL 17503-012 Gibco/Invitrogen Alterio et al., 1990; Clegg etal., 1987; Bottenstein 1981, 1988; Bottenstein et al., 1988; Morrow etal., 1990; Gonzalez et al., 1990; Moore et al., 1991; Anderson et al.,1991; Olwin et al., 1992 6 VEGF _(165 r Human) 10 μg P2654Gibco/Invitrogen Arsic et al., 2004; Germani et al., 2003; Lee et al.,2003; Lescaudron et al., 1999 7 Acidic FGF 12.5 μg 13241-013Gibco/Invitrogen Alterio et al., 1990; Moore et al., 1991; Olwin et al.,1992; Motamed et al., 2003; Dusterhoft et al., 1999; Fu et al., 1995;Smith et al., 1994; Oliver et al., 1992; Dell'Era et al., 2003 8 HeparinSulphate 50 μg D9809 Sigma Alterio et al., 1990; Moore et al., 1991;Olwin et al., 1992; Motamed et al., 2003; Dusterhoft et al., 1999; Fu etal., 1995; Smith et al., 1994; Oliver et al., 1992; Dell'Era et al.,2003 9 LIF 10 μg L5158 Sigma Husmann et al., 1996; Kurek et al., 1996;Megeney et al., 1996; Vakakis et al., 1995; Martinou et al., 1992; Sunet al., 2007; Malm et al., 2004; Zorzano et al., 2003; Sakuma et al.,2000 10 Vitronectin (Rat Plasma) 50 μg V0132 Sigma Biesecker 1990;Gullberg et al., 1995 11 CNTF 20 μg CRC 401B Cell Sciences Wang et al.,2008; Chen et al., 2003, 2005; Cannon 1998; Marques et al., 1997 12 NT 310 μg CRN 500B Cell Sciences Oakley et al., 1997 13 NT 4 10 μg CRN 501BCell Sciences Carrasco et al., 2003; Simon et al., 2003 14 GDNF 10 μgCRG 400B Cell Sciences Choi-Lundberg et al., 1995; Lin et al., 1993;Yang et al., 2004; Golden et al., 1999; Henderson et al., 1994 15 BDNF10 μg CRB 600B Cell Sciences Simon et al., 2003; Heinrich 2003; Mousaviet al., 2004 16 CT-1 10 μg CRC 700B Cell Sciences Chen et al., 2004;Bordet et al. 2001; Dolcet et al., 2001; Lesbordes et al., 2002;Nishikawa et al., 2005; Mitsumoto et al., 2001; Oppenheim et al., 2001;Peroulakis et al., 2000; Sheng et al., 1996

TABLE 2 Composition of Medium 2 No. Component(s) Amount Catalogue SourceReferences 1 Neurobasal A 500 mL 10888 Invitrogen/Gibco Brewer et al.,1993 2 Glutamax 5 mL 35050-061 Invitrogen/Gibco 3 Antibiotic-Antimycotic5 mL 15240-062 Invitrogen/Gibco 4 B27 Supplement 10 mL 17504-044Invitrogen/Gibco Das et al., 2004; Brewer et al., 1993 5 Cholesterol(250X) 5 mL 12531 Invitrogen/Gibco Jaworska-Wilczynska et al., 2002 6TNF-alpha, human 10 μg T6674 Sigma-Aldrich Caratsch et al., 1994;Al-Shanti et al., 2008; Miller et al., 1988 7 PDGF BB 50 μg P4056Sigma-Aldrich Husmann et al., 1996; Jin et al., 1991; Kudla et al.,1995; Quinn et al., 1990; Yablonka-Reuveni et al., 1995 8 Vasoactiveintestinal peptide (VIP) 250 μg V6130 Sigma-Aldrich Gold 1982 9Insulin-like growth factor 1 25 μg 12656 Sigma-Aldrich Malm et al.,2004; Zorzano et al., 2003; Al- Shanti et al., 2008 10 NAP 1 mg 61170AnaSpec. Inc. Gozes et al., 2004; Aracil et al., 2004 11 RecombinantApolipoprotein E2 50 μg P2002 Panvera Robertson et al., 2000 12 Laminin,mouse purified 2 mg 08-125 Millipore Langen et al., 2003; Foster et al.,1987; Hantai et al., 1991; Kuhl et al., 1986; Lyles et al., 1992; Songet al., 1992; Swasdison et al., 1992 13 Beta amyloid (1-40) 1 mg AG966Millipore Wang et al., 2005; Yang et al., 2007; Akaaboune et al., 200014 Human Tenascin-C protein 100 μg CC065 Millipore Hall et al., 2000 15rr-Sonic hedgehog, Shh N-terminal 50 μg 1314-SH R&D Systems Fan et al.,1994; Munsterberg et al., 1995; Nelson et al., 1996; Cossu et al., 1996;Currie et al., 1996; Norris et al., 2000; Brand-Saberi et al., 2005;Elia et al., 2007; Pagan et al., 1996; Bren-Mattison et al., 2002; Mayeset al., 2007; Koleva et al., 2005 16 rr (Agrin C terminal) 50 μg550-AG-100 R&D Systems Bandi et al., 2008; Sanes 1997Rat Embryonic Motoneuron Isolation and Co-Culture

Rat spinal motoneurons were purified from ventral cords of embryonic day14 (E14) embryos. Briefly, rats were anaesthetized and killed byinhalation of an excess of CO₂. This procedure was in agreement with theAnimal Research Council of University of Central Florida, which adheresto IACUC policies. Ventral spinal cord cells from the embryo werecollected in cold Hibernate E/GlutaMAX/antibiotic-antimycotic/B27. Thecells were dissociated with 0.05% trypsin-EDTA (Invitrogen) treatmentfor 15 min. The dissociated cells were layered over a 4 mL step gradientOptiprep diluted 0.505:0.495 (v/v) with HibernateE/GlutaMAX/antibiotic-antimycotic/B27 and then made to 15%, 20%, 25% and35% (v/v) in Hibernate E/GlutaMAX/antibiotic-anti-mycotic/B27 followedby centrifugation for 15 min, using 200 g at 4° C. After centrifugation,four bands of cells were obtained. The motoneurons with large somasconstituted the uppermost band. These cells present in the uppermostband were collected in fresh HibernateE/GlutaMAX/antibiotic-anti-mycotic/B27 and centrifuged for 5 min at 200g and 4° C. The pelleted motoneurons were resuspended in plating mediumthen plated on top of muscle cells at a density of 100 cells/mm².Motoneuron plating was performed 30 min after plating of the musclecells.

Immunocytochemistry

Neonatal Myosin Heavy Chain (Neonatal MHC)

Coverslips were rinsed with PBS, fixed in 20° C. methanol for 5-7 min,washed in PBS, incubated in PBS supplemented with 1% BSA and 0.05%saponin (permeabilization solution), and blocked for 30 min in apermeabilization solution+10% goat serum (blocking solution). Cells wereincubated overnight with primary antibody against neonatal MHC(N3.36,IgG, Developmental Studies Hybridoma Bank) diluted (1:5) in the blockingsolution. Cells were washed with PBS and incubated with AlexaFluorsecondary antibody (Invitrogen) (diluted in PBS) for 2 h. The secondaryantibody solution was removed and the cells were rinsed using PBS. Thecoverslips were dried and mounted on glass slides using VectaShield+DAPImounting medium (Vector Laboratories H-1200) and viewed on a confocalmicroscope (UltraVIEW LCI, PerkinElmer).

Double Staining with Neurofilament 150 and Neonatal Myosin Heavy Chain

Co-cultures were processed for immunocytochemistry as described above.Next, cells were incubated overnight at 4° C. with rabbitanti-neurofilament M polyclonal antibody, 150 kD, (Chemicon, AB1981,diluted 1:2000) and neonatal MHC(N3.36, IgG, Developmental StudiesHybridoma Bank diluted 1:5). After overnight incubation, the coverslipswere rinsed three times with PBS and then incubated with the AlexaFluorsecondary antibodies (Invitrogen) for 2 h. After rinsing three times inPBS, the coverslips were mounted with Vectashield+DAPI mounting mediumonto glass slides. The coverslips were visualized and images collectedusing a confocal microscope (UltraVIEW LCI, PerkinElmer). Controlswithout primary antibody were negative.

AChR+Synaptophysin Co-Staining

AChRs were labeled as described previously by incubating cultures with5×10⁻⁸ M of α-bungarotoxin, Alexa Fluor® 488 conjugate (MolecularProbes, B-13422) for 1.5 h at 37° C. before observation (Das et al.,2007 (Neuroscience)). Labeled cultures were fixed with glacial aceticacid and ethanol, washed with PBS, dried, mounted and examined byconfocal microscopy. The coverslips which were used for double stainingwith AChR+synaptophysin for locating the NMJs were processed further.After 1.5 h of α-bungarotoxin labeling of the AChR receptors, thecoverslips were fixed, blocked, permeabilized and incubated overnightwith synaptophysin antibody (MAB368, diluted 1:1000;Millipore/Chemicon), the pre-synaptic marker present in motoneuronaxonal terminals.

Data Analysis

Statistics were calculated using the following procedure. One coverslipwas randomly selected from each experiment (typically, there are sixcoverslips per experiment). 25 non-overlapping fields of view were usedto characterize each coverslip. At the magnification used, 25 fieldscovers over 40% of the surface area of the coverslip.

Results

DETA Surface Modification and Characterization

Static contact angle and XPS analysis were used for the validation ofthe surface modifications and for monitoring the quality of thesurfaces. Stable contact angles (40.64°±2.9/mean±SD) throughout thestudy indicated high reproducibility and quality of the DETA surfacesand these characteristics were similar to previously published results(Das et al., 2004; Das et al., 2007 (Nat. Protocols); Das et al., 2007(Neuroscience); Das et al., 2006; Das et al., 2003). Based on the ratioof the N is (401 and 399 eV) and the Si 2p_(3/2) peaks, XPS measurementsindicated that a reaction-site limited monolayer of DETA was formed onthe coverslips (Stenger et al., 1992).

Temporal Growth Factor Application

The formation of the maximal number of neuromuscular junctions wasobserved using the temporal growth factor application techniquedescribed in FIG. 1. Upon plating of the motoneurons and skeletalmuscle, the cells were treated with medium containing factors thatpromoted both growth and survival as well as enhancement of NMJformation (Table 1, Table 2). After 3 days in culture, the entire mediumwas removed and switched to a minimal formulation, NbActiv4, whichfacilitated both long-term survival and further development of the NMJs(FIG. 1). Further, three-fourths of the NbActiv4 medium per well wasremoved and replaced with an equal volume of fresh NbActiv4 medium. Whencompared to the continuous application of growth factors, the timedapplication resulted in cultures that lasted for up to 7 weeks asopposed to 10-12 days.

Culture Morphology of Motoneuron and Skeletal Muscle MyotubeInteractions

Phase contrast microscopy was used to visualize motoneuron axonsappearing to interact with skeletal muscle myotubes between days 12-15(FIG. 2, A-D). Some of the axonal processes appear to branch andterminate on the myotubes. Furthermore, many of the myotubes exhibitedcharacteristic striation patterns observed after sarcomere formationwhen the fibers reached approximately 25-30 days in culture (FIG. 3,A-D). Quantification of the appearance of striations after this timeindicated that the co-cultures contained about twice the number ofmyotubes showing striations.

Immunocytochemical Characterization of Motoneuron and Skeletal MuscleCo-Culture

The characteristic protein expression patterns of the motoneurons andmyotubes in co-culture were evaluated at day 25. Immunocytochemistry wasused to visualize the neurofilament protein expression in themotoneurons and neonatal myosin heavy chain (MHC) expression for themyotubes (FIG. 4, A-B). Motoneuron processes were clearly indicatedinteracting with the skeletal muscle myotubes. A band/I band formationwas more visible in the myotubes after staining with the neonatal myosinheavy chain antibody. The immunocytochemical analysis supported themorphological analysis, which had indicated the presence of striationsin double the number of myotubes as observed with the muscle onlycontrols.

Neuromuscular Junction Formation

In order to determine neuromuscular junction formation using this novelmedium formulation, the clustering of AChRs using alpha-bungarotoxin andtheir colocalization with synaptophysin vesicles was analyzedimmunocytochemically. The colocalization of these two synaptic markersindicated the proximity of pre-synaptic and post-synaptic structures andwas a positive indication of NMJ formation. This technique was used toidentify the colocalization of synaptophysin vesicles with the AChRclusters (FIG. 5, A-D). The axon+myotube interactions that did notresult in the colocalization of pre-synaptic and post-synapticstructures were also identified (FIG. 6, A-B). The observation of thenegative result defines the difference between colocalization andnon-colocalization and emphasizes the positive result observed in thissystem. FIG. 7 illustrates NMJ formation between a myotube in culturethat did not stain for neonatal myosin heavy chain and a motoneuron.

Discussion

This work documents the substantial improvement of an in vitro modelsystem for NMJ formation. Specifically, we observed enhancedsurvivability of the culture resulting in our ability to conductlong-term studies on the motoneuron-skeletal muscle cocultures. Thisincreased survivability resulted in maturation of the skeletal musclemyotubes and a significant improvement in the number of NMJs formed inculture.

Previously, we developed the first defined culture model to cocultureembryonic motoneuron and fetal skeletal muscle, however this model wasnot suitable for long-term tissue engineering studies and the myotubesin the culture only expressed an early muscle marker, i.e. fetal myosinheavy chain and none of the myotubes exhibited characteristicstriations. In this study, significant improvement over our previousmotoneuron-skeletal muscle co-culture model system was documented. Thisnew culture model supported long-term co-culture of both motoneuron andmuscle, resulted in a more adult-like morphology of the muscle and ahigher density of neuromuscular junctions (NMJ). Our findings weresupported by morphological and immunocytochemical data.

We developed this serum-free medium, supplemented with growth factorsthat supported the survival, proliferation and fusion of fetal ratmyoblasts into contractile myotubes, in a semi-empirical fashion. Therationale for selecting the growth factors was based on the distributionof their cognate receptors in the developing myotubes in rat fetus(Arnold et al., 1998; Brand-Saberi et al., 2005; Olson et al., 1992).Tables 1 and 2 reference the literature where these individual growthfactors, hormones and neurotransmitters were observed to support muscleand neuromuscular junction development. The composition in Table 1 isthe formulation used for a previously published medium utilized formotoneuron-muscle co-culture and adult spinal cord neuron culture (Daset al., 2007 (Neuroscience); Das et al., 2008 (Exp. Neurology); Das etal., 2005; Das et al., 2007 (Biomaterials)). Table 2 lists the twelveadditional factors identified in muscle development and neuromuscularjunction formation that enabled the increased survivability of thesystem. Further addition of the factors in Table 2 promoted formation ofcharacteristic striation in the muscle in the culture. The use ofNbActiv4 for the maintenance of the cells significantly improved thesurvival of the skeletal muscle derived myotubes despite the fact thatthe original purpose of the development of NbActiv4 was for thelong-term maintenance and synaptic connectivity of fetal hippocampalneurons in vitro (Brewer et al., 2008).

In our previous co-culture model, we did not observe the expression ofneonatal MHC proteins in the myotubes. Interestingly, when this samemedium and protocol was used to culture pure skeletal muscle we observedcertain striking differences. The pure muscle culture survived longer,exhibited characteristic striations, but only a very small percentage ofmyotubes expressed N3.36 (Das et al., 2009 (Biomaterials)). To the bestof our understanding, the N3.36 expression in skeletal muscle in cultureis influenced by the motoneurons either physically or by certain trophicfactors secreted in the presence of this modified medium and NbActiv4.This observation needs further studies in order to dissect the molecularpathways regulating N3.36 expression in pure skeletal muscle culture andin skeletal muscle-motoneuron co-culture. Also, the potential regulationof MHC class switching independent of neuronal innervation/denervationrepresents an interesting topic for further study. This system wouldhave applications in developing therapies for muscle-nerve diseases suchas ALS, spinal muscular atrophy, spinal cord injury and myastheniagravis.

CONCLUSIONS

The development of robust NMJ formation, long-term survival ofmotoneuron

skeletal muscle co-cultures and selective MHC class switching isdocumented in this research. This improved system supports the goal ofcreating a physiologically relevant tissue engineered motoneuron

skeletal muscle construct and puts within reach the goal of developingfunctional bio-hybrid devices to analyze NMJ activity. This definedmodel can also be used to map the developmental pathways regulating NMJformation and MHC class switching. Furthermore, we believe this serumfree culture system will be a powerful tool in developing advancedstrategies for regenerative medicine in ALS, stretch reflex arcdevelopment and integrating motoneuron+skeletal muscle with bio-hybridprosthetic devices. Due to the use of a serum-free defined culturesystem this also has applications for new high-throughput screeningsystems for use in drug discovery research and toxicologyinvestigations.

Accordingly, in the drawings and specification there have been disclosedtypical preferred embodiments of the invention and although specificterms may have been employed, the terms are used in a descriptive senseonly and not for purposes of limitation. The invention has beendescribed in considerable detail with specific reference to theseillustrated embodiments. It will be apparent, however, that variousmodifications and changes can be made within the spirit and scope of theinvention as described in the foregoing specification and as defined inthe appended claims.

REFERENCES CITED

-   [1] Akaaboune M, et al. (2000) Developmental regulation of amyloid    precursor protein at the neuromuscular junction in mouse skeletal    muscle. Mol Cell Neurosci. 15(4):355-367.-   [2] Al-Shanti N, et al. (2008) Beneficial synergistic interactions    of TNF•alpha and IL•6 in C2 skeletal myoblasts—potential cross-talk    with IGF system. Growth Factors. 26(2):61-73.-   [3] Alterio J, et al. (1990) Acidic and basic fibroblast growth    factor mRNAs are expressed by skeletal muscle satellite cells.    Biochem Biophys Res Commun. 166(3):1205-1212.-   [4] Anderson J E, et al. (1991) Distinctive patterns of basic    fibroblast growth factor (bFGF) distribution in degenerating and    regenerating areas of dystrophic (mdx) striated muscles. Dev Biol.    147(1):96-109.-   [5] Aracil A, et al. (2004) Proceedings of Neuropeptides 2004, the    XIV European Neuropeptides Club meeting. Neuropeptides.    38(6):369-371.-   [6] Arnold H H, et al. (1998) more complexity to the network of    myogenic regulators. Curr Opin Genet Dev. 8(5):539-44.-   [7] Arsic N, et al. (2004) Vascular endothelial growth factor    stimulates skeletal muscle regeneration in vivo. Mol. Ther.    10(5):844-854.-   [8] Bandi E, et al. (2008) Neural agrin controls maturation of the    excitation-contraction coupling mechanism in human myotubes    developing in vitro. Am J Physiol Cell Physiol. 294(1):C66-73.-   [9] Biesecker G, et al. (1990) The complement SC5b•9 complex    mediates cell adhesion through a vitronectin receptor. J. Immunol.    145(1):209-214.-   [10] Bordet T., et al. (2001) Protective effects of cardiotrophin-1    adenoviral gene transfer on neuromuscular degeneration in transgenic    ALS mice. Hum Mol. Genet. 10(18):1925-1933.-   [11] Bottenstein J E, et al. (1988) CNS neuronal cell line-derived    factors regulate gliogenesis in neonatal rat brain cultures. J    Neurosci Res. 20(3):291-303.-   [12] Bottenstein J E, et al. (1998) Advances in vertebrate cell    culture methods. Science. 239(4841 Pt 2):G42, G48.-   [13] Bottenstein J E, et al. (1981) Proliferation of glioma cells in    serum-free defined medium. Cancer Treat Rep. 65 Suppl 2:67-70.-   [14] Brand-Saberi B, et al. (2005) Genetic and epigenetic control of    skeletal muscle development. Ann Anat. 187(3):199-207.-   [15] Bren-Mattison Y, et al. (2002) Sonic hedgehog inhibits the    terminal differentiation of limb myoblasts committed to the slow    muscle lineage. Dev Biol. 242(2):130-148.-   [16] Brewer G J, et al. (2008) NbActiv4 medium improvement to    Neurobasal/B27 increases neuron synapse densities and network spike    rates on multielectrode arrays. J Neurosci Methods. 170(2):181-187.-   [17] Brewer G J, et al. (1993) Optimized survival of hippocampal    neurons in B27-supplemented Neurobasal, a new serum-free medium    combination. J Neurosci Res. 35(5):567-576.-   [18] Cannon J G, et al. (1998) Intrinsic and extrinsic factors in    muscle aging Ann NY Acad Sci. 854:72-77.-   [19] Caratsch C G, et al. (1994) Interferon-alpha, beta and tumor    necrosis factor-alpha enhance the frequency of miniature end-plate    potentials at rat neuromuscular junction. Neurosci Lett.    166(1):97-100.-   [20] Carrasco Dl, et al. (2003) Neurotrophin 4/5 is required for the    normal development of the slow muscle fiber phenotype in the rat    soleus. J Exp Bio. 206(Pt 13):2191-2200.-   [21] Chen J, et al. (2004) Role of exogenous and endogenous trophic    factors in the regulation of extraocular muscle strength during    development. Invest Ophthalmol V is Sci. 45(10):3538-3545.-   [22] Chen X, et al. (2005) Dedifferentiation of adult human    myoblasts induced by ciliary neurotrophic factor in vitro. Mol Bioi    Cell. 16(7):3140-3151.-   [23] Chen X P, et al. (2003) [Exogenous rhCNTF inhibits myoblast    differentiation of skeletal muscle of adult human in vitro]. Sheng    Li Xue Bao. 55(4):464-468.-   [24] Choi-Lundberg D L, et al. (1995) Ontogeny and distribution of    glial cell line-derived neurotrophic factor (GDNF) mRNA in rat.    Brain Res Dev Brain Res. 85(1):80-88.-   [25] Chow I, et al. (1985) Release of acetylcholine from embryonic    neurons upon contact with muscle cell. J. Neurosci. 5(4):1076-82.-   [26] Ciegg C H, et al. (1987) Growth factor control of skeletal    muscle differentiation: commitment to terminal differentiation    occurs in G1 phase and is repressed by fibroblast growth factor. J.    Cell Biol. 105(2):949-956.-   [27] Colomar A, et al. (2004) Glial modulation of synaptic    transmission at the neuromuscular junction. Glia. 47(3):284-9.-   [28] Cossu G, et al. (1996) How is myogenesis initiated in the    embryo? Trends Genet. 12(6):218-223.-   [29] Currie P D, et al. (1996) Induction of a specific muscle cell    type by a hedgehog-like protein in zebrafish. Nature.    382(6590):452-455.-   [30] Daniels M P, et al. (2000) Rodent nerve-muscle cell culture    system for studies of neuromuscular junction development:    refinements and applications. Microsc Res Tech. 49(1):26-37.-   [31] Daniels M P, (1997) Intercellular communication that mediates    formation of the neuromuscular junction. Mol. Neurobiol.    14(3):143-170.-   [32] Das M, et al. (2008) Temporal neurotransmitter conditioning    restores the functional activity of adult spinal cord neurons in    long-term culture. Exp Neurol. 209(1): 171-180.-   [33] Das M, et al. (2005) Adult rat spinal cord culture on an    organosilane surface in a novel serum-free medium. In Vitro Cell Dev    Bioi Anim. 41(10):343-348.-   [34] Das M, et al. (2003) Electrophysiological and morphological    characterization of rat embryonic motoneurons in a defined system.    Biotechnol Prog. 19(6):1756-1761.-   [35] Das M, et al. (2004) Long-term culture of embryonic rat    cardiomyocytes on an organosilane surface in a serum-free medium.    Biomaterials. 25(25):5643-5647.-   [36] Das M, et al. (2007) Auto-catalytic ceria nanoparticles offer    neuroprotection to adult rat spinal cord neurons. Biomaterials.    28(10): 1918-1925.-   [37] Das M, et al. (2009) Skeletal muscle tissue engineering: an    improved model promoting long term survival of myotubes, structural    development of e-c coupling apparatus and neonatal myosin heavy    chain (MHC) expression. Biomaterials. 30:5392-402.-   [38] Das M, et al. (2007) Embryonic motoneuron-skeletal muscle    co-culture in a defined system. Neuroscience. 146(2):481-488.-   [39] Das M, et al. (2007) Differentiation of skeletal muscle and    integration of myotubes with silicon microstructures using    serum-free medium and a synthetic silane substrate. Nat. Protoc.    2(7): 1795-1801.-   [40] Das M, et al. (2006) A defined system to allow skeletal muscle    differentiation and subsequent integration with silicon    microstructures. Biomaterials. 27(24):437 4-4380.-   [41] Dell'Era P, et al. (2003) Fibroblast growth factor receptor 1    is essential for in vitro cardiomyocyte development. Circ Res.    93(5):414-420.-   [42] Dolcet X, et al. (2001) Cytokines promote motoneuron survival    through the Janus kinase-dependent activation of the    phosphatidylinositol 3-kinase pathway. Mol Cell Neurosci.    18(6):619-631.-   [43] Dusterhoft S, et al. (1999) Evidence that acidic fibroblast    growth factor promotes maturation of rat satellite-cell-derived    myotubes in vitro. Differentiation. 65(3): 161-169.-   [44] Eisenberg T, et al. (2009) Induction of autophagy by spermidine    promotes longevity. Nat Cell Biol. 11(11):1305-14.-   [45] Elia D, et al. (2007) Sonic hedgehog promotes proliferation and    differentiation of adult muscle cells: Involvement of MAPK/ERK and    P13K/Akt pathways. Biochim Biophys Acta. 1773(9):1438-1446.-   [46] English A W., et al. (2003) Cytokines, growth factors and    sprouting at the neuromuscular junction. J. Neurocytol.    32(5):943-60.-   [47] Fan C M, et al. (1994) Patterning of mammalian somites by    surface ectoderm and notochord: evidence for sclerotome induction by    a hedgehog homolog. Cell. 79(7):1175-1186.-   [48] Foster R F, et al. (1987) A laminin substrate promotes    myogenesis in rat skeletal muscle cultures: analysis of replication    and development using antidesmin and anti-BrdUrd monoclonal    antibodies. Dev Bio. 122(1):11-20.-   [49] Fu X, et al. (1995) Acidic fibroblast growth factor reduces rat    skeletal muscle damage caused by ischemia and reperfusion. Chin Med    J (Engl). 108(3):209-214.-   [50] Germani A, et al. (2003) Vascular endothelial growth factor    modulates skeletal myoblast function. Am J. Pathol.    163(4):1417-1428.-   [51] Gold M R, et al. (1982) The effects of vasoactive intestinal    peptide on neuromuscular transmission in the frog. J. Physiol.    327:325-335.-   [52] Golden J P, et al. (1999) Expression of neurturin, GDNF, and    GDNF family-receptor mRNA in the developing and mature mouse. Exp    Neural. 158(2):504-528.-   [53] Gonzalez A M, et al. (1990) Distribution of basic fibroblast    growth factor in the 18-day rat fetus: localization in the basement    membranes of diverse tissues. J. Cell Biol. 110(3):753-765.-   [54] Gozes I, et al. (2004) NAP mechanisms of neuroprotection. J    Mol. Neurosci. 24(1):67-72.-   [55] Gullberg D, et al. (1995) Analysis of fibronectin and    vitronectin receptors on human fetal skeletal muscle cells upon    differentiation. Exp Cell Res. 220(1):112-123.-   [56] Hall B K, et al. (2000) All for one and one for all:    condensations and the initiation of skeletal development. Bioessays.    22(2): 138-147.-   [57] Hantai D, et al. (1991) Developmental appearance of    thrombospondin in neonatal mouse skeletal muscle. Eur J. Cell Biol.    55(2):286-294.-   [58] Heinrich G, et al. (2003) A novel BDNF gene promoter directs    expression to skeletal muscle. BMC Neurosci. 4:11.-   [59] Henderson C E, et al. (1994) GDNF: a potent survival factor for    motoneurons present in peripheral nerve and muscle. Science.    266(5187):1062-1064.-   [60] Husmann I, et al. (1996) Growth factors in skeletal muscle    regeneration. Cytokine Growth Factor Rev. 7(3):249-258.-   [61] Jaworska-Wilczynska M, et al. (2002) Three lipoprotein    receptors and cholesterol in inclusion-body myositis muscle.    Neurology. 58(3):438-445.-   [62] Jin P, et al. (1991) Recombinant platelet-derived growth    factor-88 stimulates growth and inhibits differentiation of rat L6    myoblasts. J Biol. Chem. 266(2):1245-1249.-   [63] Koleva M, et al. (2005) Pleiotropic effects of sonic hedgehog    on muscle satellite cells. Cell Mol Life Sci. 62(16): 1863-1870.-   [64] Kudla A J, et al. (1995) A requirement for fibroblast growth    factor in regulation of skeletal muscle growth and differentiation    cannot be replaced by activation of platelet-derived growth factor    signaling pathways. Mol Cell Biol. 15(6):3238-3246.-   [65] Kuhl U, et al. (1986) Role of laminin and fibronectin in    selecting myogenic versus fibrogenic cells from skeletal muscle    cells in vitro. Dev Biol. 117(2):628-635.-   [66] Kurek J B, et al. (1996) Leukemia inhibitory factor and    interleukin-6 are produced by diseased and regenerating skeletal    muscle. Muscle Nerve. 19(10):1291-1301.-   [67] Langen R C, et al. (2003) Enhanced myogenic differentiation by    extracellular matrix is regulated at the early stages of myogenesis.    In Vitro Cell Dev Bioi Anim. 39(3-4): 163-169.-   [68] Lee E W., et al. (2003) Neuropeptide Y induces ischemic    angiogenesis and restores function of ischemic skeletal muscles. J    Clin Invest. 111(12): 1853-1862.-   [69] Lesbordes J C, et al. (2002) In vivo electrotransfer of the    cardiotrophin-1 gene into skeletal muscle slows down progression of    motor neuron degeneration in pmn mice. Hum Mol. Genet.    11(14):1615-1625.-   [70] Lescaudron L, et al. (1999) Blood borne macrophages are    essential for the triggering of muscle regeneration following muscle    transplant. Neuromuscul Disord. 9(2):72-80.-   [71] Li M X, et al. (2001) Opposing actions of protein kinase A and    C mediate Hebbian synaptic plasticity. Nat. Neurosci. 4(9):871-872.-   [72] Lin L F, et al. (1993) GDNF: a glial cell line-derived    neurotrophic factor for midbrain dopaminergic neurons. Science.    260(5111):1130-1132.-   [73] Lyles J M, et al. (1992) Matrigel enhances myotube development    in a serum-free defined medium. Int J Dev Neurosci. 10(1):59-73.-   [74] Malm C, et al. (2004) Leukocytes, cytokines, growth factors and    hormones in human skeletal muscle and blood after uphill or downhill    running J. Physiol. 556(Pt 3):983-1000.-   [75] Marques M J, et al. (1997) Ciliary neurotrophic factor    stimulates in vivo myotube formation in mice. Neurosci Lett.    234(1):43-46.-   [76] Martinou J C, et al. (1992) Cholinergic differentiation factor    (CDF/LIF) promotes survival of isolated rat embryonic motoneurons in    vitro. Neuron. 8(4):737-744.-   [77] Maves L, et al. (2007) Pbx homeodomain proteins direct Myod    activity to promote fast-muscle differentiation. Development.    134(18):3371-3382.-   [78] Megeney L A, et al. (1996) bFGF and LIF signaling activates    STAT3 in proliferating myoblasts. Dev Genet. 19(2):139-145.-   [79] Miller S C, et al. (1998) Tumor necrosis factor inhibits human    myogenesis in vitro. Mol Cell Biol. 8(6):2295-2301.-   [80] Mitsumoto H, et al. (2001) Effects of cardiotrophin•1 (CT-1) in    a mouse motor neuron disease. Muscle Nerve. 24(6):769-777.-   [81] Moore J W, et al. (1991) The mRNAs encoding acidic FGF, basic    FGF and FGF receptor are coordinately downregulated during myogenic    differentiation. Development. 111(3):741-748.-   [82] Morrow N G, et al. (1990) Increased expression of fibroblast    growth factors in a rabbit skeletal muscle model of exercise    conditioning. J Clin Invest. 85(6):1816-1820.-   [83] Motamed K, et al. (2003) Fibroblast growth factor receptor-1    mediates the inhibition of endothelial cell proliferation and the    promotion of skeletal myoblast differentiation by SPARC: a role for    protein kinase A. J Cell Biochem. 90(2):408-423.-   [84] Mousavi K, et al. (2004) BDNF rescues myosin heavy chain 118    muscle fibers after neonatal nerve injury. Am J Physiol Cell    Physicl. 287(1):C22-29.-   [85] Munsterberg A E, et al. (1995) Combinatorial signaling by Sonic    hedgehog and Wnt family members induces myogenic bHLH gene    expression in the somite. Genes Dev. 9(23):2911-2922.-   [86] Nelson C E, et al. (1996) Analysis of Hox gene expression in    the chick limb bud. Development. 122(5):1449-1466.-   [87] Nelson P G (1975) Nerve and muscle cells in culture. Physiol    Rev. 55(1):1-61.-   [88] Nishikawa J, et al. (2005) Increase of Cardiotrophin-1    immunoreactivity in regenerating and overloaded but not denervated    muscles of rats. Neuropathology. 25(1): 54-65.-   [89] Norris W, et al. (2000) Slow muscle induction by Hedgehog    signalling in vitro. J Cell Sci. 113 (Pt 15):2695-2703.-   [90] Oliver L, et al. (1992) Acidic fibroblast growth factor (aFGF)    in developing normal and dystrophic (mdx) mouse muscles.    Distribution in degenerating and regenerating mdx myofibres. Growth    Factors. 7(2):97-106.-   [91] Oakley R A, et al. (1997) Neurotrophin-3 promotes the    differentiation of muscle spindle afferents in the absence of    peripheral targets. J. Neurosci. 17(11):4262-4274.-   [92] Olson E N., et al. (1992) Interplay between proliferation and    differentiation within the myogenic lineage. Dev Biol.    154(2):261-72.-   [93] Olwin B B, et al. (1992) Repression of myogenic differentiation    by aFGF, bFGF, and K•FGF is dependent on cellular heparan    sulfate. J. Cell Biol. 118(3):631-639.-   [94] Oppenheim R W, et al. (2001) Cardiotrophin-1, a muscle-derived    cytokine, is required for the survival of subpopulations of    developing motoneurons. J. Neurosci. 21(4):1283-1291.-   [95] Pagan S M, et al. (1996) Surgical removal of limb bud Sonic    hedgehog results in posterior skeletal defects. Dev Biol.    180(1):35-40.-   [96] Peroulakis M E, et al. (2000) Forger N G: Ciliary neurotrophic    factor increases muscle fiber number in the developing levator ani    muscle of female rats. Neurosci Lett. 296(2-3):73-76.-   [97] Quinn L S, et al. (1990) Paracrine control of myoblast    proliferation and differentiation by fibroblasts. Dev Biol.    140(1):8-19.-   [98] Ravenscroft M S, et al. (1998) Developmental Neurobiology    Implications from Fabrication and Analysis of Hippocampal Neuronal    Networks on Patterned Silane-Modified Surfaces. J Am Chem. Soc.    120(47):12169-12177.-   [99] Robertson T A, et al. (2000) Comparison of astrocytic and    myocytic metabolic dysregulation in apolipoprotein E deficient and    human apolipoprotein E transgenic mice. Neuroscience. 98(2):353-359.-   [100] Sakuma K, et al. (2000) Differential adaptation of growth and    differentiation factor 8/myostatin, fibroblast growth factor 6 and    leukemia inhibitory factor in overloaded, regenerating and    denervated rat muscles. Biochim Biophys Acta. 1497(1):77-88.-   [101] Sandi E, et al. (2008) Neural agrin controls maturation of the    excitation-contraction coupling mechanism in human myotubes    developing in vitro. Am J Physiol, Cell Physiol. 294(1):C66-73.-   [102] Sanes J R, et al. (1997) Genetic analysis of postsynaptic    differentiation at the vertebrate neuromuscular junction. Curr Opin    Neurobiol. 7(1):93-100.-   [103] Sheng Z, et al. (1996) Cardiotrophin-1 displays early    expression in the murine heart tube and promotes cardiac myocyte    survival. Development. 122(2):419-428.-   [104] Simon M, et al. (2003) Effect of NT-4 and BDNF delivery to    damaged sciatic nerves on phenotypic recovery of fast and slow    muscles fibres. Eur J. Neurosci. 18(9):2460-2466.-   [105] Smith J, et al. (1994) The effects of fibroblast growth    factors in long-term primary culture of dystrophic (mdx) mouse    muscle myoblasts. Exp Cell Res. 210(1):86-93.-   [106] Song W K, et al. (1992) H36-alpha 7 is a novel integrin alpha    chain that is developmentally regulated during skeletal    myogenesis. J. Cell Biol. 117(3):643-657.-   [107] Stenger D A, et al. (1992) Coplanar molecular assemblies of    aminoalkylsilane and perfluorinated alkylsilane—characterization and    geometric definition of mammalian-cell adhesion and growth. J Am    Chem. Soc. 114(22):8435-42.-   [108] Sun L, et al. (2007) JAK1-STAT1-STAT3, a key pathway promoting    proliferation and preventing premature differentiation of    myoblasts. J. Cell Biol. 179(1): 129-138.-   [109] Swasdison S, et al. (1992) Formation of highly organized    skeletal muscle fibers in vitro. Comparison with muscle development    in vivo. J Cell Sci. 102 (Pt 3):643-652.-   [110] Torgan C E, et al. (2006) Calcineurin localization in skeletal    muscle offers insights into potential new targets. J Histochem    Cytochem. 54(1): 119-128.-   [111] Torgan C E, et al. (2001) Regulation of myosin heavy chain    expression during rat skeletal muscle development in vitro. Mol Bioi    Cell. 12(5): 1499-1508.-   [112] Vakakis N, et al. (1995) In vitro myoblast to myotube    transformations in the presence of leukemia inhibitory factor.    Neurochem Int. 27(4-5):329-335.-   [113] Vogel Z, et al. (1976) Ultrastructure of acetylcholine    receptor clusters on cultured muscle fibers. J. Cell Biol.    69(2):501-507.-   [114] Wang P, et al. (2005) Defective neuromuscular synapses in mice    lacking amyloid precursor protein (APP) and APP-Like protein 2. J.    Neurosci. 25(5): 1219-1225.-   [115] Wang X, et al. (2008) Effects of interleukin-6, leukemia    inhibitory factor, and ciliary neurotrophic factor on the    proliferation and differentiation of adult human myoblasts. Cell    Mol. Neurobiol. 28(1): 113-124.-   [116] Witzemann V., (2006) Development of the neuromuscular    junction. Cell Tissue Res. 326(2):263-71.-   [117] Yablonka-Reuveni Z, et al. (1995) Development and postnatal    regulation of adult myoblasts. Microsc Res Tech. 30(5):366-380.-   [118] Yang L, et al. (2007) Increased asynchronous release and    aberrant calcium channel activation in amyloid precursor protein    deficient neuromuscular synapses. Neuroscience. 149(4):768-778.-   [119] Yang L X, et al. (2004) Glia cell line-derived neurotrophic    factor regulates the distribution of acetylcholine receptors in    mouse primary skeletal muscle cells. Neuroscience. 128(3):497-509.-   [120] Zorzano A, et al. (2003) Intracellular signals involved in the    effects of insulin-like growth factors and neuregulins on myofibre    formation. Cell Signal. 15(2):141-149.

That which is claimed:
 1. A method of co-culturing mammalian musclecells and motoneurons, the method comprising: suspending fetal musclecells and fetal spinal motoneurons in a serum-free medium according tocomposition 1 of Table 1; placing the suspended fetal muscle cells andfetal spinal motoneurons onto a monolayer of covalently bondedtrimethoxysilylpropyl-diethylenetriamine supported on an underlyingcarrier surface; covering the carrier comprising muscle cells andmotoneurons in a mixture of serum-free medium composition 1 of Table 1and serum-free medium composition 2 of Table 2; and incubating thecovered carrier comprising muscle cells and motoneurons.
 2. The methodof claim 1, wherein the fetal muscle cells and motoneurons originatefrom fetal rats.
 3. The method of claim 1, wherein the underlyingcarrier surface comprises a glass cover slip.
 4. The method of claim 1,wherein incubating is under mammalian physiological conditions.
 5. Themethod of claim 1, wherein incubating is at approximately 37° C. in anatmosphere of air with about 5% CO₂ and 85% humidity.
 6. The method ofclaim 1, wherein the mixture of medium composition 1 of Table 1 andmedium composition 2 of Table 2 comprises approximately equal volumes ofeach composition.
 7. The method of claim 1, further comprising changingthe covering medium to a Neurobasal/B27/Glutamax-based medium comprisingcreatine, estrogen, and cholesterol without growth factors as incubatingproceeds.
 8. The method of claim 1, further comprising changing thecovering medium to a Neurobasal/B27/Glutamax-based medium comprisingcreatine, estrogen, and cholesterol without growth factors during thefirst week of incubation.
 9. The method of claim 8, further comprisingperiodically changing the covering medium after the first week withfresh Neurobasal/B27/Glutamax-based medium comprising creatine,estrogen, and cholesterol without growth factors.
 10. The method ofclaim 1, further comprising, during the incubating, monitoring thecarrier for formation of myotubes by the incubated muscle cells.
 11. Themethod of claim 1, further comprising, during the incubating, monitoringthe carrier for formation of neuromuscular junctions between theincubated motoneurons and muscle cells.